Tuberculosis (TB) caused by the slow growing bacillus Mycobacterium tuberculosis (Mtb) is the leading cause of infectious disease mortality in the world by a bacterial pathogen. The mycobactins have been proposed as novel targets for TB drugs since these small-molecule iron-chelators (siderophores) produced by Mycobacterium tuberculosis (MTb) are responsible for obtaining iron from the human host, a process that is essential for the survival of MTb. Additionally, the mycobactins may serve as a short-term iron reservoir in Mtb. Inhibition of mycobactin biosynthesis is expected to block iron acquisition and potentially disrupt iron homeostasis. We propose to develop a new class of antibacterial agents that target siderophore biosynthesis. The primary focus of this application will be on the organism Mycobacterium tuberculosis; however, the Gram-negative Acinetobacter baumannii and Klebsiella pneumoniae will also be pursued. In the first specific aim we will build on our substantial knowledge of the structure activity relationships of our lead compound 5'-O-[N-(salicyl)sulfamoyl]adenosine (Sal-AMS) to improve drug disposition properties. Compounds will be investigated to determine pharmacokinetic properties and then evaluated in an in vivo model of infection using a murine model of TB. Additionally, we will explore 1) new analogues to confirm our hypothesized binding model, 2) analogues with an improved spectrum of antibacterial activity, and 3) a new series of nonnucleoside inhibitors identified from high-throughput screening. In the second specific aim, pharmacokinetic studies will be performed and compounds evaluated in a murine TB model. Newly synthesized analogues will also be assayed for enzyme inhibition, antibacterial activity, and drug disposition properties. In a final subaim, we propose to perform mechanism of action studies to identify potential off-target receptors targeted by our prototypical siderophore inhibitors. In the third specific aim, we propose to synthesize transitions-state inhibitors of a new enzyme, which catalyzes the first biosynthetic step in production of the siderophores from M. tuberculosis. These rationally designed inhibitors will be evaluated for enzyme inhibition, co-crystallized with the molecular target, evaluated for antitubercular activity and toxicity, and finally their mechanism of action will be explored using whole-cell radioassays. It is expected that upon completion of this we will have validated our hypothesis that siderophore-mediated iron acquisition is essential in vivo. Thus, the research proposed herein is expected to have a positive impact on human health and may additionally validate a new class of antibiotics that target siderophore biosynthesis. PUBLIC HEALTH RELEVANCE: Tuberculosis (TB) caused by the slow growing bacillus Mycobacterium tuberculosis (Mtb) is the leading cause of infectious disease mortality in the world by a bacterial pathogen. M. tuberculosis and other atypical mycobacteria are now classified as opportunistic infections of AIDS patients. The proposed research is expected to validate siderophore biosynthesis as new antibacterial target, which may lead to the development of a new class of antitubercular agents.